The proposed research is based on the observation that the cellular protein CEM15 suppresses infectivity of human immunodeficiency virus type-1 (HIV-1) that lacks the virion infectivity factor, Vif. CEM15 is homologous to the mammalian mRNA editing enzyme APOBEC-1. It is hypothesized that CEM15 suppresses HIV-1 infectivity as a direct consequence of its deaminase activity on viral or host cell nucleic acids and that Vif inhibits this activity. Specific Aim 1 will use structural modeling of CEM15 to predict sitespecific mutants that will address the relationship between cytidine deaminase activity and anti-viral activity. These studies will be accomplished utilizing in vitro deaminase assays that will include Vif in order to assess its ability to inhibit deaminase activity. Viral infectivity will be quantified using a novel vif + and vif- HIV-1 pseudotyped lentiviral particle assay that is amenable to the rapid demarcation of regions of HIV-1 targeted by CEM15. Specific Aim 2 will determine the viral DNA or RNA substrate(s) of CEM15 and determine the effect of Vif expression on substrate utilization. Construction of the corresponding site-specific, HIV-1 mutants and quantifying their infectivity relative to wild type HIV-1 will determine the biological significance of the observed dC to dU changes. Emphasis will also be placed on the analysis of potential of C to U modification of the cellular tRNAlys3 primer that initiates viral reverse transcription. Additionally, a bacterial DNA mismatch detection system wherein DNA repair confers a positive selection for clones containing CEM15 modifications will be employed to identify sites of C to U modification in viral or cellular RNA or DNA isolated from infected cells. This research focuses on the role of CEM15's deaminase activity and target substrate in the suppression of HIV infectivity. Moreover, the structural modeling of CEM15 will aid in the identification of potential Vif binding sites and localization of anti-viral activity to specific segments of CEM15, which will be instrumental in development of a new category of anti-HIV-1 therapeutics.